Project Summary The development of functional neurons requires extensive polarized membrane traffic, to drive axonal and dendritic tree extension and organize synaptic connections. An essential factor in the delivery of proteins to the plasma membrane is the exocyst, which is a complex of 8 subunits that functions to tether vesicles to the membrane prior to their fusion. Exocyst accumulates in the growth cone during neurite outgrowth of hippocampal neurons, and in puncta along the axon prior to synaptogenesis. Disruption of an exocyst gene in Drosophila blocks neurite outgrowth, although neurotransmitter release persists. Functional exocyst is required for viability in all organisms tested. Remarkably, however, human patients have recently been found with mutations in an exocyst subunit (EXOC2) that cause severe neurological defects including micro- or macrocephaly and mental retardation. Nothing is known about structural deficits in the mutant exocyst. Indeed, the structure of the mammalian exocyst complex remains unknown, and the dynamics of assembly/disassembly and vesicle tethering are still not fully understood. In this project we will develop powerful tools for structural and live cell analysis of the exocyst in primary neurons, and to investigate molecular mechanisms underlying the neurological defects caused by exocyst subunit mutation. First, we will create new knock-in mouse lines that express fluorophore-tagged exocyst subunits, using CRISPR strategy to generate GFP-Exo70 and Halo-Sec8. (We have shown that gene-edited murine cell lines expressing tagged endogenous exocyst subunits are viable and the exocyst is functional). These mice will enable for the first time the analysis of exocyst protein dynamics in primary neurons. Embryonic cortical neurons and brain slices will be used to investigate exocyst expression, localization and dynamics during neurite extension and synaptogenesis. Second, we will edit the human EXOC2 mutation into the mouse genome. We will use homozygotes, if viable, to investigate the molecular defect that causes the neurological phenotype in patients. If the mutant is late embryonic lethal, we will isolate primary neurons from embryos and use brain slices to image exocyst localization and measure vesicle docking and fusion efficiencies. If early embryonic lethal we will create a floxed mutant strain, to enable conditional expression of the mutation in the developing brain, using a nestin Cre. Finally, the new mouse strains described above will provide sufficient purified mammalian exocyst for cryo-EM and other structural studies. Exocyst complexes will be purified from sfGFP-exocyst brain tissue using GFP nanobody beads. Mass spectrometry will determine the purity of the complex and identify associated accessory proteins under different purification conditions. Biochemical studies will investigate the stoichiometry and stability of the WT and mutant complex; and cryo-EM imaging will test the feasibility of deriving, for the first time, high resolution structures of the mammalian exocyst.